Methods of measuring amount of cholesteryl ester in a blood sample

ABSTRACT

A method of measuring an amount of at least one cholesteryl ester in a blood sample is carried out by combining a blood sample with a salt and an alcohol to provide a dilute sample; introducing the dilute sample into a mass spectrometer; and then measuring the amount of the at least one cholesteryl ester in the dilute sample by mass spectrometry, to thereby measure the amount of at least one cholesteryl ester in the blood sample, is described.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national phase entry of PCTApplication PCT/US2012/039999, filed May 30, 2012, and published inEnglish on Dec. 6, 2012, as International Publication No. WO2012/166798, and which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application Ser. No. 61/492,083, filed Jun. 1,2011, the disclosure of each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention concerns devices, systems, computer-based methodsand methods for generating a prediction of risk and/or indication ofextent of coronary stenosis or atherosclerosis in human subjects, fordiagnostic purposes in subjects who are afflicted with chest pain orsymptoms concerning for acute coronary syndrome, as well as forprognostic purposes in subjects who are not afflicted with chest pain.

BACKGROUND OF THE INVENTION

Annually in the US, 6 million patients present to emergency departmentswith acute chest pain or related complaints.(1) Most patients with acutechest pain presenting to emergency departments undergo an evaluation forpossible acute coronary syndrome (ACS), yet the vast majority does nothave this disease process. After exclusion of myocardial infarction (MI)with serial cardiac necrosis biomarkers, practice guidelines recommendfurther evaluation of patients with possible ACS with stress testing orcoronary CT angiography (CCTA).(2) This practice is currently necessaryto prevent the discharge of patients with unstable angina, but leads toa large number of negative tests. A biomarker is needed to predictpatients likely to have underlying coronary artery disease (CAD) amongpatients with acute chest pain. A biomarker better able to identifypatients likely to have coronary disease could improve testingefficiency by either reducing pretest probability below the testingthreshold, or by guiding selection of the cardiac imaging modality.

Traditional cardiac risk factors, such as smoking, diabetes, andhypercholesterolemia, have been shown to correlate strongly with thelong-term risk of developing coronary atherosclerosis.(3-5) In patientswith acute chest pain, these risk factors have been shown to be onlyweak predictors of cardiac chest pain.(6) The rationale for thisapparent discrepancy relates to the importance of the clinical historyand the presence of acute chest pain being a much stronger relativepredictor for symptomatic disease than these traditional risk factors.

SUMMARY OF THE INVENTION

The present invention provides systems and methods for determining aprediction of risk and/or an indication of extent of coronary stenosisin a human subject. The method comprises the steps of: providing orinputting a level of at least one cholesteryl ester measured (e.g., bymass spectroscopy) in a blood sample collected from the subject; andthen determining from the cholesteryl ester level a prediction of riskand/or indication of extent of coronary stenosis in the subject.

In some embodiments, the method further comprises the step of: providingthe age and/or gender of the subject; and wherein the determining stepincludes determining a prediction of risk and/or indication of extent ofcoronary stenosis in the subject from the cholesteryl ester level, alongwith the age and/or gender of the subject.

In some embodiments, the subject is afflicted with acute chest pain(angina) or other symptom of acute coronary syndrome; in someembodiments, the coronary stenosis is significant coronary stenosis.

In some embodiments, the subject is not afflicted with acute chest painor other symptom of acute coronary syndrome; in some embodiments, thecoronary stenosis is moderate coronary stenosis.

In some embodiments, the cholesteryl ester comprises, consists of orconsists essentially of CE 16:1 and CE 18:1 in combination.

Recently, acyl-CoA:cholesterol acyltransferase-2 (ACAT2) activity hasbeen shown in monkey and murine models to correlate withatherosclerosis.(7-9) Hepatic ACAT2 is the primary source of cholesterylester (CE) produced from the mono-unsaturated fatty acids palmitoleicacid (16:1) and oleic acid (18:1) and has also been associated with CEproduced from palmitic acid (16:0). Higher plasma concentrations ofthese CE have been linked to increased risk for MI in a longitudinalcohort of men followed over 19 years.(10) Whether measurement of theseCE in plasma may be informative to care providers when assessingpatients with acute chest pain has heretofore been unclear.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of a non-limiting example of a system andmethod of the invention.

FIG. 2. CE values by the presence of significant coronary stenosis. Theboxplots represent the values of the sum of cholesteryl esters 16:1 and18:1 measured in participants. The box margins represent the 25^(th) and75^(th) percentiles, the bar within the box represents the median, andthe whiskers represent the range of values.

FIG. 3. Receiver operator characteristic curves for full (CE, age,gender, number of conventional risk factors) and reduced (full modelminus CE) models to predict any CAD among the entire study cohort. Thefull model has a significantly higher C statistic, p=0.0219.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention now will be described more fully hereinafter withreference to the accompanying figures, in which embodiments of theinvention are shown. This invention may, however, be embodied in manyalternate forms and should not be construed as limited to theembodiments set forth herein. Like numbers refer to like elementsthroughout the description of the figures.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The present invention may be embodied as systems, methods, and/orcomputer program products. Accordingly, the present invention may beembodied in hardware and/or in software (including firmware, residentsoftware, micro-code, etc.). Furthermore, the present invention may takethe form of a computer program product on a computer-usable orcomputer-readable storage medium having computer-usable orcomputer-readable program code embodied in the medium for use by or inconnection with an instruction execution system. In the context of thisdocument, a computer-usable or computer-readable medium may be anynon-transient medium that can contain or store the program for use by orin connection with the instruction execution system, apparatus, ordevice.

The computer-usable or computer-readable medium may be, for example butnot limited to, an electronic, magnetic, electromagnetic, infrared, orsemiconductor system, apparatus, or device. More specific examples (anonexhaustive list) of the computer-readable medium would include thefollowing: an electrical connection having one or more wires, a portablecomputer diskette, a random access memory (RAM), a read-only memory(ROM), an erasable programmable read-only memory (EPROM or Flashmemory), and a portable compact disc read-only memory (CD-ROM).

1. DEFINITIONS

“Cholesteryl ester” as used herein refers to an ester of cholesterol andone or more fatty acids. The ester bond is formed between thecarboxylate group of the fatty acid and the hydroxyl group ofcholesterol. Examples of particular cholesteryl esters that may bemeasured to carry out the present invention include, but are not limitedto, cholesteryl myristate (CE 14:0), cholesteryl palmitate (CE 16:0),cholesteryl palmitoleate (CE 16:1), cholesteryl heptadecenoate (CE17:1), cholesteryl stearate (CE 18:0), cholesteryl oleate (CE 18:1),cholesteryl linoleate (CE 18:2), cholesteryl linolenate (CE 18:3)(including cholesteryl alpha-linolenate (CE 18:3n-3) and cholesterylgamma-linolenate (CE 18:3n-6)), cholesteryl arachidonate (CE 20:4),cholesteryl eicosatrienoate (CE 20:3), cholesteryl eicosapentaenoate (CE20:5), cholesteryl docosapentaenoate (CE 22:5), cholesteryldocosahexaenoate (CE 22:6), and combinations thereof (including anycombination of 2, 3, or 4 or more thereof).

“Risk of coronary stenosis” as used herein is different from “extent ofcoronary stenosis” as discussed below, and is intended to convey aprobability that a subject is afflicted with coronary stenosissufficient to warrant further diagnostic, preventative, therapeutic,and/or interventional medical attention. Risk may be conveyed in avariety of ways, including but not limited to: a scale such as 1 to 10;as a risk category (low vs. moderate vs. high), as a binaryclassification (at risk vs. not at risk), as a percent likelihood). Ifdesired, the indication of risk may further comprise, or be in the formof, an indication of extent of coronary stenosis in the subject.

“Extent of coronary stenosis” refers to the severity of coronarystenosis and/or the burden of coronary atherosclerosis and as usedherein may be indicated in any suitable manner. The indication may begiven as a verbal or text explanation (e.g., serious coronary stenosis,versus significant coronary stenosis, versus moderate coronary stenosis,versus minor coronary stenosis, or any combination or subcombinationthereof) with suitable definitions of terms as necessary. The indicationmay be given as a range (e.g., a scale of 1 to 5 or 10, with 1indicating little stenosis and 5 or 10 indicating severe stenosis), as apercentage of occlusion (including ranges thereof), as a color warningin a visual display (e.g., green, for little or no stenosis, yellow formoderate coronary stenosis; red for significant coronary stenosis),and/or by any other suitable technique.

“Significant coronary stenosis” as used herein refers to a blockage offifty percent or more of a named coronary artery (for example, the rightcoronary artery, the posterior descending artery, the right marginalartery, the left coronary artery, the left circumflex artery, the leftmarginal artery, and the left anterior descending artery (including thediagonal branch).

“Moderate coronary stenosis” as used herein refers to detectablecoronary stenosis in any coronary artery, but with less than a fiftypercent blockage of any named coronary artery.

“Blood sample” as used herein may be any suitable blood sample,including blood plasma, blood serum, and whole blood.

“Risk factor” or “additional risk factor” as used herein includes, butis not limited to, hyperlipidemia, smoking, diabetes, hypertension,obesity, substance abuse (e.g., alcohol abuse or alcoholism), sedentarylifestyle or lack of exercise, family history including race (e.g., oneor more parent with heart disease), etc. Age (e.g., over 65) and gender(particularly male gender) may be considered risk factors as well, butare addressed separately herein.

“Subject” as used herein are human subjects of any race and gender.While the subjects may be of any age, including juvenile and adolescentsubjects, typical subjects are adult and geriatric subjects. Subjectsmay be with, or without, chest pain (angina) or other symptoms of acutecoronary syndrome.

“Symptoms of acute coronary syndrome” include, but are not limited to:chest pain (angina), particularly chest pain that feels like burning,pressure, or tightness and lasts several minutes or longer; painelsewhere in the body such as the left upper arm or jaw; nausea;vomiting; shortness of breath; sudden, heavy sweating, etc.

2. ELEMENTS OF THE INVENTION

The present invention can be carried out by any suitable technique.Whole blood can be collected from a subject, processed if desired intoblood serum or plasma, and further prepared for measurement ofcholesteryl esters therein by any suitable technique, including knowntechniques or the mass spectrometry techniques described below. Once ameasurement of cholesteryl esters in a patient sample is obtained, anindication of risk and/or extent of coronary stenosis can be determinedby reference to a table or tables generated by the data and/ortechniques described herein. Separate tables and/or table entries can beprovided based on the subject's age and/or gender, and the presence orabsence of other risk factors (such as those discussed below) ifdesired.

In some embodiments, the invention may be carried out as acomputer-based method for determining a prediction of risk and/or anindication of extent of coronary stenosis in a human subject, asillustrated in FIG. 1. Such methods typically comprise: (a) inputtingthe level of at least one cholesteryl ester measured in a blood samplecollected from the subject; (b) inputting the age and gender of thesubject (which can be done before or after inputting step (a)); and then(c) generating from the cholesteryl ester level, the age and the gendera prediction of risk and/or an indication of extent of coronary stenosisin the subject. The generating step may be carried out by any suitabletechnique, such as with a regression model or an empirically-based modelof actual clinical experience.

In some embodiments, the method may further comprise inputting thepresence or absence of at least one additional risk factor (and in someembodiments 2 or 3 or more additional risk factors) in the subject.Hence, the generating step further comprises generating from thecholesteryl ester level, the age, and the at least one additional riskfactor a prediction of risk and/or indication of extent of coronarystenosis in the subject.

In some embodiments, such as where the subject is afflicted with acutechest pain or other indication of acute coronary syndrome, theindication of coronary stenosis can be an indication of significantcoronary stenosis. In other embodiments, such as where the subject isnot afflicted with acute chest pain or other indication of acutecoronary syndrome, the indication of coronary stenosis may be anindication of moderate coronary stenosis.

In some embodiments, the cholesteryl ester measured comprises, consistsof or consists essentially of CE 16:1 and CE 18:1, in combination (e.g.,the sum of the two thereof).

3. MASS SPECTROSCOPY

The level of the at least one cholesteryl ester in the blood sample canbe determined by any suitable means, but in some embodiments ispreferably determined by mass spectrometry.

Thus, the present invention provides a method of measuring the amount ofat least one cholesteryl ester in a blood sample by mass spectrometry.The method comprises:

-   -   combining (in any convenient sequence or order and at any        suitable temperature such as room temperature) the blood sample        with a salt and a diluents such as an alcohol (and optionally        other diluents or ingredients such as water, buffering agents,        etc.) to provide a dilute sample;    -   introducing the dilute sample into a mass spectrometer (e.g., by        directly introducing the sample into the mass spectrometer,        typically by introducing or injecting a volume of from 0.1 or 1        to 100 or 500 microliters thereof); and then    -   measuring the amount of the at least one cholesteryl ester in        the dilute sample by mass spectrometry (i.e., in the mass        spectrometer into which the sample has been introduced) to        thereby measure (and quantify) the amount of at least one        cholesteryl ester in the blood sample.        If desired, an additional known cholesteryl ester (typically        different from the CE's being measured) may be added to the        dilute sample in a known amount as an internal standard. While        particular conditions for performing the steps, such as times        and temperatures, are not critical, in some preferred        embodiments, all of the steps are carried out within a        relatively short period of time: e.g., within 24 hours, within 8        hours, or in some embodiments in 2 hours or less.

In some embodiments, the diluent is provided in an amount sufficient toachieve at least a 50, 100 or 200-fold (by volume) dilution of the bloodsample, up to a 1000 or 2000-fold dilution (or more). For example, insome embodiments, the blood sample is included in the diluents in anamount ranging from 0.01 percent by volume, up to 1, 2, or 10 percent byvolume. Suitable alcohols for use as diluents include, but are notlimited to, methanol, ethanol, and isopropanol. Without wishing to belimited to any theory of the invention, the diluent may be included inan amount sufficient to solubilize the at least one cholesteryl ester inthe sample.

In some embodiments, the salt is added in an amount of from 0.001 or0.01 mg to 1 or 10 mg of the salt in the dilute sample. Suitable saltsinclude, but are not limited to, sodium formate, sodium acetate,ammonium formate, ammonium acetate, lithium formate, lithium acetate,potassium formate, potassium acetate, and other alkali metal salts offatty acids. Without wishing to be limited to any theory of theinvention, the salt may be included in an amount sufficient to form ametal adduct with the cholesteryl ester in the sample.

Suitable equipment for carrying out mass spectrometry is known andspecific techniques for carrying out the methods of the invention can becarried out in accordance with known procedures or variations thereofthat will be apparent to those skilled in the art. See, e.g., E. deHoffman and V. Stroobant, Mass Spectrometry: Principles and Applications(3d ed. 2007).

The present invention is explained in greater detail in the non-limitingexamples set forth below.

Experimental

The objective of this study is to test whether there is potentialdiagnostic value in measuring plasma levels of ACAT2 associated CEs whenevaluating patients with possible ACS. This investigation examineswhether there is any association between measured CE levels and anydefinable coronary stenosis, or significant coronary stenosis, measuredat angiography. The premise is that a more accurate estimate of apatient's likelihood of having CAD, when presenting with acute chestpain, will improve healthcare delivery by allowing more judicious use ofdiagnostic tests.

I. Methods.

Study Design.

The study, a single site observational cohort, was approved by theinstitutional review board of the Wake Forest University School ofMedicine and all participants provided written informed consent.

Study Setting and Population.

The study population consisted of patients at the study institution whowere at least 18 years old undergoing angiography of the coronaryarteries with either cardiac catheterization or computed tomography. Theoverall goal of the study was to assemble a repository for the testingof novel cardiac biomarkers; therefore, only patients with inability tofollow up or anemia (hemoglobin <8.0) were excluded. The observationalcohort consisted of two distinct patient populations from Wake ForestUniversity Baptist Medical Center, patients referred for invasivecoronary angiography, and patients undergoing coronary computedtomographic angiography (CCTA) in the observation unit of the emergencydepartment. The analysis of plasma CE composition to use as a biomarkerwas the a priori primary analysis from this cohort.

This analysis includes patients enrolled from either the ED or thehospital setting and further required that the patients had chest painor related symptoms. The ED population consists of patients whopresented with chest pain or related complaints, received a primaryassessment, and the care providers felt them be at low risk for ACSbased on initial cardiac biomarkers and the initial electrocardiogram.After the primary assessment, the patients were placed in the EDobservation unit, had a CCTA ordered, and were then approached forenrollment. The basis for the clinical assessment was an overallimpression of low risk based on the framework set forth in the ACC/AHAguidelines(11) and a Thrombolysis in Myocardial Infarction (TIMI) riskscore(12) of 0 or 1 correlating to a short term risk for ACS of 2-5%(13). Patients recruited from the hospital setting were patients withand without known ACS being referred to angiography for eitherdefinition of coronary anatomy or coronary intervention.

Study Procedures.

After obtaining informed consent, participants underwent a single blooddraw, provided background information about their history anddemographics, and completed a food frequency questionnaire. The foodfrequency questionnaire administered was the Block Brief 2000(NutritionQuest, Berkeley, Calif.), chosen for both its brevity and theability to quantify monounsaturated, polyunsaturated, and saturated fatintake. Clinical outcomes at 30 days were assessed using a scriptedtelephone interview and a structured medical record review. The primaryinvestigator reviewed all case report forms to make a finaldetermination of ACS during the index hospital visit and at 30 daysbased on an objective study definition and blinded to CE measurements.Clinical data collection was consistent with standardized guidelines forED patients with possible ACS.(14)

Coronary Imaging.

Participants undergoing coronary angiography had findings abstractedfrom the clinical reports. Invasive angiography was performed usingconventional techniques and the most severe diameter stenosis wasrecorded on the case report form for each reported vessel. Participantswith prior bypass grafts had stenoses recorded for both native and graftvessels. Methods used at the study institution to assess the coronaryvasculature with CCTA have been previously described (ref in press). Inbrief, all participants underwent coronary imaging with a 64-sliceLightSpeed VCT (GE Healthcare, Milwaukee, Wis.). Most patients firstreceived oral and/or IV beta-blockade. Initial scout images wereobtained followed by a low-dose, noncontrast, ECG-gated acquisition forcalcium scoring. The subsequent contrast injection was a triple-phaseintravenous injection consisting of 100 mL of nonionic iodinatedcontrast (Optiray 350; Mallinckrodt Medical, Hazelwood, Mo.) followed byan ECG-gated acquisition. Raw image data was then used to createmultiphase images that were post-processed and analyzed on anindependent 3D workstation (Advantage Workstation 4.2, GE Healthcare).Image interpretation occurred as part of clinical care. Interpretingradiologists and cardiologists meeting level 2 or 3 training guidelinesfrom the ACC/AHA for cardiac CT.(15) Coronary stenosis was measuredusing an electronic caliper and comparing the average luminal diameterin the most stenotic region with the average luminal diameter of anormal proximal or distal reference segment located within 1 cm of thestenosis without intervening branch vessels.

Data Handling and Follow Up.

Sources of data included the participants, care providers, and themedical record. Data templates were used to collect data directly fromthe patient and care providers for data fields that were anticipated tobe unreliable in the medical record. Abstraction of medical record datawas guided by a “sources of data” document describing the expectedlocation and definition of each data field. Data from paper case reportforms were then entered into a web-based electronic database.

Sample Acquisition and Processing.

Whole blood was collected via venipuncture into vacutainer tubescontaining EDTA. Cells were separated from plasma by centrifugation at aminimum of 1700 RPM for 15 min in a tabletop centrifuge at 4° C. Plasmawas subsequently aspirated from the cell layer. Plasma was stored at−70° C. until analysis was done. Lipoprotein cholesterol distributionswere determined on whole plasma using size separations via gelfiltration chromatography, similar to the method of Garber, et al.(16)Plasma (containing about 15 μg of cholesterol) diluted 1:1 with coldphosphate buffered saline was applied to a Superose 6 column (GEHealthcare). The column is eluted with 0.9% saline containing 0.01% EDTAand 0.01% sodium azide at a flow rate of 0.4 ml/min using a LaChromEliteHPLC system (Hitachi High Technologies) and the column eluate iscontinuously mixed online with 0.125 ml/min cholesterol reagent(Cholesterol Liquid Reagent Set, Pointe Scientific, Inc) that is thenpassed through a 5 mL knitted reaction coil maintained at 37° C. Datareadout is proportional to cholesterol concentration in the eluate, andfractions containing VLDL, LDL and HDL are identified so that thepercentage of cholesterol in each can be determined. The concentrationsin each lipoprotein class are then calculated from a direct measure ofcholesterol concentration in an aliquot of the starting plasma. Themeasurement of cholesterol and triglyceride concentrations in wholeplasma was done using enzymatic methods for cholesterol(17) andtriglycerides(18).

Mass spectrometry was used for measurement of CEs. Samples were storedat −70° C. before analysis. After thawing, 5 μL of plasma was added to 1mL methanol solution containing 5 ng/μL 17:0 CE (internal standard) and10 ng/μL sodium formate. The solution was vortexed for 10 seconds andthen allowed to stand at room temperature for 30 minutes. 100 μL of thefirst solution was diluted 1:10 with high purity methanol. IndividualCEs were measured using a Quattro II mass spectrometer equipped with aZ-spray interface. Analysis parameters were as follows: capillaryvoltage=3.2 kV, cone voltage=50V, source temperature=80° C., anddesolvation temperature=200° C. Samples were maintained at 15° C. in atemperature controlled Spark Holland Reliance autosampler/stacker whileawaiting analysis. 25 μL of each sample was infused into the massspectrometer at 10 μL/min. CE were quantified in the positive ion modeby monitoring the common neutral loss of 368.4 Da. The CE profile andquantitation were calculated from this data and presented in anelectronic spreadsheet. Cholesteryl esters measured included those withthe following fatty acids: palmitate, 16:0; palmitoleate, 16:1;stearate, 18:0; oleate, 18:1; linoleate, 18:2; linolenate, 18:3;arachidonate, 20:4; eicosapentenoate, 20:5; and docosahexanoate, 22:6.Measurements were reported in percentages of total CE by mass and plasmaconcentrations (10⁻³ mol/m³) of each, and both measures were examined inthe data analysis.

Primary Outcomes.

The primary outcome was the presence of CAD, measured either by CCTA orinvasive angiography. Severity of the maximal diameter of stenosis wasrecorded and the patients were dichotomized as a) positive or negativefor CAD, and b) positive or negative for significant CAD (≧50%). Rangesof stenosis were recorded as the highest value of the range. Stenosiscaused by myocardial bridging alone was not included in this analysis.

Sample Size.

Variance and effect size data for this novel biomarker were notavailable in humans. At the outset it was empirically estimated thatapproximately 100 participants with varying degrees of CAD would provideat least preliminary evidence of a clinically important relationship, ifone existed. It was estimated that this would provide sufficient eventrates of coronary stenosis to satisfactorily fit exploratory multiplevariable logistic regression models, considering the standard “rule often” recommending ten events for every degree of freedom considered inthe multivariate model.²⁶

Data Analysis.

The first objective was to determine if there was an association betweenmeasured CE levels and coronary stenosis. Two definitions of coronarystenosis were examined, any measurable CAD and significant (>50%)stenosis. Means and standard deviations, medians with inter-quartileranges, and proportions were used to describe normally distributedcontinuous variables, skewed continuous variables and categoricalvariables, respectively. Cholesterol esters related to ACAT2 (16:0,16:1, and 18:1) and their sums were examined among participants with andwithout coronary stenosis and compared with Kruskal-Wallis tests.Cholesteryl palmitate concentrations contributed in a differentdirection than cholesteryl palmitoleate and cholesteryl oleate.Mechanistically, ACAT2 is directly involved in the synthesis of thelatter two cholesteryl esters after the activity of steroyl CoAdesaturase 1 to introduce the double bond, whereas the synthesis ofcholesteryl palmitate does not require prior steroyl CoA desaturase 1activity. Therefore this CE was removed and the sum of 16:1 and 18:1were further examined.

Logistic regression was used to assess the relationship between the sumof cholesteryl palmitoleate plus cholesteryl oleate and coronarystenosis. Separate models using absolute CE concentrations, masspercentages, and both CAD endpoints (any stenosis, >50% stenosis) wereconstructed. Univariate logistic regression was performed betweenpotential covariates and the endpoint under consideration, and thosecovariates with a Wald p<0.20 were then considered for inclusion in themultivariate model. Covariates included age, dietary intake ofmonounsaturated and polyunsaturated fat, gender, total plasmacholesterol, LDL, HDL, body mass index, and a summary measure oftraditional coronary risk factors (smoking, diabetes, hypertension,dyslipidemia) ranging from zero to four. Graphic examination andfractional polynomial analysis(19) indicated that continuous variableswere best treated as linear within the models. Model construction wasperformed manually beginning with the full model with backward removalof covariates in order to build a parsimonious model. The CE term wasretained at all steps; a Wald p<0.05 was required for other covariatesto be retained within the model. Interaction terms were generated viathe product method and assessed for inclusion, as were covariates withthe potential to be effect modifiers of the relationship between the CEand endpoint. Additional models were generated to assess theserelationships within the low risk ED subgroup with acute chest pain. Anadditional set of models examined the sum of CE concentrations (16:1,18:1, 18:3, 20:4, 20:5, and 22:6) as the primary predictor covariateagainst the outcomes of any stenosis and significant stenosis. Given theexploratory nature of this analysis, we made no adjustment to any pvalues for the construction of multiple models. The Hosmer-Lemeshowgoodness of fit test was used to assess model fit, and receiver areaoperating curves were generated to produce a c-statistic. Contributionsof CE were assessed between the final full model and a reduced modelwithout the CE by comparison of the receiver operator characteristic(ROC) curve C statistic, and integrated discrimination improvement(IDI). The IDI is a measure of the discriminatory ability of the modelswith and without the marker of interest. Each model was assessed foroutliers and overly influential points utilizing Pregibon's dbetastatistic. Additional models were generated to assess theserelationships within the low risk ED subgroup with acute chest pain. Anadditional set of models examined the sum of CE concentrations (16:1,18:1, 18:3, 20:4, 20:5, and 22:6) as the primary predictor covariateagainst the outcomes of any stenosis and significant stenosis.

The secondary objective was to examine the potential effect of theresulting final model in the subgroup of ED patients with low risk chestpain. This was first accomplished by comparing the final model with theDiamond and Forrester classification using the IDI. The final model wasalso then applied to the low risk subgroup to determine the potentialreduction in imaging and the resulting missed ACS rate at 30 days. Dataanalysis was conducted with SAS Enterprise Guide, v4.2 (SAS InstituteInc., Cary, N.C.) and Stata/IC 11.2 (College Station, Tex.).

II. Results

The observational cohort consisted of 150 participants enrolled over 24months at the time of this analysis. Of these 150, 37 participants wereexcluded for meeting one or more of the following exclusions: noavailable blood samples (n=10), no coronary imaging (n=7), no foodfrequency questionnaire data (n=21), and no acute chest pain (n=3). Thefinal dataset for this analysis consisted of 113 participants withcomplete data; 58% were enrolled from the observation unit and 42% wereenrolled from the angiography suite.

Participants were a mean age of 49 (+/−11.7) years, 38% were women(Table 1). Conventional coronary risk factors among study participantsincluded hypertension (43%), current smoking (37%), diabetes (19%),hyperlipidemia (42%), history of cocaine use (16%), and 16% of the studypopulation had experienced a prior MI. Most participants had a chiefcomplaint of chest pain (91%) and a normal (53%) or nonspecific ECG(28%) nearest to enrollment. During the index visit, 23% had experiencedMI and 26% had received revascularization (Table 2).

TABLE 1 Participant Demographics and Past Medical History No CAD CADPatient Characteristics n/N (%) n/N (%) Age (years)* 40.9 (8.6) 55.0(10.0) Female sex 23/46 (50.0) 20/67 (29.9) White race 28/46 (60.9)53/67 (79.1) Hypertension 12/46 (26.1) 36/67 (53.7) Diabetes mellitus4/46 (8.7) 17/67 (25.4) Current smoking 17 (37.0) 25/67 (37.3) Historyof cocaine use 8/46 (17.4) 10/67 (14.9) Hyperlipidemia (by history) 9/46(19.6) 38/67 (56.7) Body Mass Index* 29.7 (5.8) 29.8 (5.8) Prior HeartFailure 0/46 (0) 3/67 (4.5) Prior myocardial infarction 1/46 (2.2) 17/67(25.4) *= data presented as mean(SD); CABG = Coronary artery bypassgraft;

TABLE 2 Presenting Characteristics and Physical Exam Findings No CAD CADn/N (%) n/N (%) Presenting Characteristics Chest pain chief complaint39/44 (88.6) 61/66 (92.4) Chest pain at rest 36/43 (83.7) 45/65 (69.2)Multiple episodes of symptoms within 15/43 (34.9) 29/64 (45.3) 24 hoursof presentation Chest pain pleuritic 8/43 (18.6) 12/63 (19.0) PhysicalExam Heart rate (beats/minute)* 78.9 (14.4) 68.9 (23.1) Systolic bloodpressure (mmHg)* 136.6 (35.4) 131.3 (31.6) Murmur 1/42 (2.4) 6/65 (9.2)Rales 0/44 (0) 6/65 (9.2) Jugular venous distention 0/43 (0) 0/65 (0)Overall electrocardiogram classification Normal 30/46 (65.2) 30/67(44.8) Nonspecific changes 13/46 (28.3) 19/67 (28.4) Earlyrepolarization only 0/46 (0) 2/67 (3.0) Abnormal but not diagnostic of1/46 (2.2) 2/67 (3.0) ischemia Infarction or ischemia known to be old 0(0) 4/67 (6.0) Infarction or ischemia not known to be 1/46 (2.2) 7/67(10.5) old Suggestive of myocardial infarction 1/46 (2.2) 3/67 (4.5)Risk Stratification TIMI risk score 0 25/46 (54.4) 12/67 (17.9) 1 17/46(37.0) 16/67 (23.9) 2 3/46 (6.5) 11/67 (16.4) 3 1/46 (2.2) 15/67 (22.4)4 0/46 (0) 10/67 (14.9) 5 0/46 (0) 2/67 (3.0) 6 0/46 (0) 1/67 (1.5)30-day acute coronary syndrome 1/46 (2.2) 35/67 (52.2) Cardiovasculardeath 0/46 (0) 1/67 (1.5) Myocardial infarction 1/46 (2.2) 25/67 (37.3)Revascularization 0/46 (0) 29/67 (43.3) *= data presented as mean(SD);TIMI = thrombolysis in myocardial infarction

Computed tomography coronary angiography was the diagnostic standard forcoronary imaging in 56% of participants with the remainder undergoinginvasive angiography. At angiography, 59% had coronary stenosis and 43%had significant coronary stenosis in at least 1 named coronary artery.Two participants undergoing CCTA had major coronary segments that couldnot be quantified due to artifact and were classified based on theavailable information. As a measure of disease burden, coronary arterydisease severity scores(20) were calculated among participants withsignificant coronary stenosis for each main coronary vessel (excludingthe 2 participants with incomplete data for this calculation). Amongthese participants, the severity score averaged across all 4 maincoronary vessels (including left main) was 4.3 (+/−6.1). At least 50%stenosis was seen in 1, 2, 3, and 4 coronary distributions in 17, 11, 9,and 1 participants, respectively.

Stratifying the cohort into participants with and without CAD, dietaryfat intake and plasma lipid concentrations were similar between groups(Table 3). Cholesteryl ester concentrations were significantly higherfor 16:1, 18:1, 18:3, 20:4, 20:5, and 22:6 among participants withcoronary stenosis (Table 4, FIG. 2). Based on the study hypothesis, thesum CE (16:1 and 18:1) was of primary interest and was significantlyhigher in both mass percent and concentration in those with CAD. FIG. 2suggests that sum CE (16:1 and 18:1) may be more useful in patients 40years old or younger. This subgroup consisted of 29 participants, 6 withany CAD.

TABLE 3 Dietary intake, serum and plasma results No CAD CAD Mean (SD)Mean (SD) p value Dietary intake (daily estimated intake) Total fat (g)79.8 (39.2) 81.3 (51.2) 0.86 Saturated fat (g) 26.5 (13.2) 26.4 (17.2)0.96 Monounsaturated 31.0 (16.1) 31.6 (19.6) 0.86 fat (g)Polyunsaturated 16.0 (7.8) 17.1 (11.7) 0.54 fat (g) Dietary 235.7(137.2) 257.0 (203.3) 0.51 cholesterol (g) Olive oil use for 23/46(50.0) 30/67 (44.8) 0.70† cooking, n/N (%) Plasma lipids (mg/dl) Totalcholesterol 166.6 (27.5) 178.7 (44.6) 0.0780 VLDL 19.7 (12.1) 23.8(14.5) 0.1237 LDL 105.0 (21.0) 113.7 (36.0) 0.1094 HDL 41.9 (13.1) 41.3(16.2) 0.83 Comparisons were conducted using t-tests. VLDL = very lowdensity lipoprotein; LDL = low density lipoprotein; HDL = high densitylipoprotein; For all analyses, p <= .05 denotes statisticalsignificance, with no adjustment for the multiple comparisons.

TABLE 4 Plasma cholesteryl ester results Median concentration in μmol/lMedian percentage of CE (Q1, Q3) (Q1, Q3) No CAD CAD p value No CAD CADp value 16:0 449.4 (375.3, 489.0) 440.4 (379.0, 547.9) 0.52 10.2 (9.4,11.1) 9.6 (8.6, 10.4) 0.0066 16:1 112.1 (85.1, 156.0) 150.5 (118.8,214.8) 0.0007 2.8 (2.2, 3.5) 3.3 (2.6, 4.2) 0.0128 18:0 58.2 (45.2,64.8) 66.9 (45.8, 85.0) 0.0641 1.3 (1.1, 1.5) 1.4 (1.0, 1.7) 0.41 18:1645.8 (568.4, 767.4) 791.4 (648.7, 939.8) 0.0016 15.8 (14.1, 17.0) 16.2(15.0, 17.7) 0.0879 18:2 2421.3 (2108.2, 2733.6) 2657.5 (2195.6, 3049.0)0.0674 56.3 (51.4, 59.2) 54.6 (51.3, 58.0) 0.0766 18:3 81.6 (64.8,106.4) 111.2 (90.3, 146.4) 0.0002 1.9 (1.6, 2.3) 2.3 (2.1, 2.6) 0.001420:4 425.6 (359.5, 515.0) 474.8 (404.7, 615.8) 0.0166 10.3 (8.9, 12.0)10.8 (9.3, 12.8) 0.2264 20:5 34.1 (27.9, 47.4) 41.2 (33.1, 62.5) 0.02560.9 (0.7, 1.1) 0.9 (0.7, 1.2) 0.50 22:6 19.8 (15.7, 26.1) 22.6 (17.5,31.1) 0.0402 0.5 (0.4, 0.6) 0.5 (0.4, 0.6) 0.32 Sum 775.5 (669.3, 937.4)945.5 (757.5, 1106.6) 0.0012 18.4 (16.0, 21.0) 19.3 (17.8, 22.1) 0.0354(16:1, 18:1) Comparisons were conducted using a Kruskal-Wallis Test; Q1= first quartile; Q3 = third quartile; CAD = coronary artery disease;For all analyses, p <= .05 denotes statistical significance, with noadjustment for the multiple comparisons.

In logistic regression modeling using participants with complete data(n=113), the absolute concentration of sum CE (16:1 and 18:1) waspredictive of any coronary stenosis in the final model also containingage, gender, and number of conventional coronary risk factors (AUC 0.95,95% CI 0.91-0.98). In this model, each 10 micromolar increase inconcentration was associated with a 6.5% increase in the odds of havingcoronary stenosis (p<0.001). No interaction terms were found to beadditive to the explanatory value of the model. The Hosmer-Lemeshowgoodness of fit test (^(X) ² ₍₈₎=13.9, p=0.08) provided no reason toreject the primary model on the basis of fit. To determine thecontribution of sum CE (16:1 and 18:1) to the model, ROC curves werecreated and are shown in FIG. 3 for the model with and without sum CE(16:1 and 18:1). The model without sum CE (16:1 and 18:1) had asignificant reduction in the C statistic to 0.89 (95% CI 0.81-0.94, pfor comparison 0.004). The estimated integrated discriminationimprovement from adding sum CE (16:1 and 18:1) was 0.15 (p<0.001)suggesting the sum CE adds to the ability of the model to discriminatebetween those with and without CAD.

The performance of the final model for CAD was then compared to theDiamond Forrester risk stratification framework in the subgroup of lowrisk observation unit patients with complete data for this calculation(n=58). When compared, the clinical model had superior performance basedon an estimated IDI (0.403, p<0.001) indicating that the final model forCAD had better discriminatory ability. In low risk patients with serialnegative troponin results (n=64), implementing a 50% predictedprobability threshold to determine the need for further testing afterserial cardiac markers would have led to a calculated maximum post-testprobability of 1% based on the prevalence of ACS after excluding MI of2%¹⁰ (2%*50%=1.0%). At this threshold, 42/64 (66%) patients would havebeen considered negative; 1 had maximal stenosis of 50%, none with30-day ACS. A 50% predicted probability threshold would have resulted in22/64 (34%) patients with positive results, 8 had maximal stenosis ≧50%and none had 30-day ACS.

Exploratory analyses demonstrated that when modeling significantcoronary stenosis, the variables in the final model were unchanged (AUC0.89). In this model, each 10 micromolar increase in concentration ofthe sum (16:1, 18:1) was associated with a 3.5% increase in the odds forhaving significant stenosis (p=0.001).

When restricting the analysis to the low risk subgroup, the sum (16:1,18:1) was similarly predictive. The final model for any coronarystenosis also contained age and gender (AUC 0.90). In this model, each10 micromolar increase in the sum (16:1, 18:1) was associated with anincrease in odds of any stenosis of 6.5% (p=0.001). The final model forsignificant coronary stenosis only included age and the predictor (AUC0.89). In this model, each 10 micromolar increase in the sum (16:1,18:1) was associated with a 4.2% increase in the odds for significantcoronary stenosis (p=0.003).

In the analysis including the sum (16:1, 18:1, 18:3, 20:4, 20:5, and22:6) as the predictor, there was no improvement in AUC of the models orthe value of the predictor to the model compared to the simpler sum(16:1, 18:1) predictor. In no models were any interaction terms found tobe significant. Models for each outcome and each set of predictors areas follows:

-   Model 1: Entire study cohort, outcome=Any coronary disease, primary    predictor=sum (16:1, 18:1)

Logistic regression Number of obs = 113 LR chi2(4) = 87.25 Prob > chi2 =0.0000 Log likelihood = −32.735808 Pseudo R2 = 0.5713 AUC .9468 Oddsany_cad Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum 1.006534.0018363 3.57 0.000 1.002941 1.010139 (16:1, 18:1) age 1.241218 .06149044.36 0.000 1.126365 1.367782 male 9.436404 7.676202 2.76 0.006 1.91594246.47621 Number 1.997805 .6365916 2.17 0.030 1.069846 3.730651 riskfactors

-   Model 2: Observation subgroup, outcome=any coronary disease, primary    predictor=sum (16:1, 18:1)

Logistic regression Number of obs = 62 LR chi2(3) = 37.09 Prob > chi2 =0.0000 Log likelihood = −21.779809 Pseudo R2 = 0.4599 auc = 0.9034 Oddsany_cad Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum 1.006468.0019305 3.36 0.001 1.002692 1.010259 (16:1, 18:1) age 1.200838 .07819862.81 0.005 1.05695 1.364315 male 4.865638 4.332211 1.78 0.076 .849682827.86268

-   Model 3: Entire study cohort, outcome=significant coronary disease,    primary predictor=sum (16:1, 18:1)

Logistic regression Number of obs = 113 LR chi2(4) = 62.96 Prob > chi2 =0.0000 Log likelihood = −45.848868 Pseudo R2 = 0.4071 AUC = 0.8938sig_cad Odds Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum 1.00354.0010197 3.48 0.001 1.001544 1.005541 (16:1, 18:1) age 1.154613 .03932284.22 0.000 1.080057 1.234314 male 3.718503 2.210141 2.21 0.027 1.15996611.92041 Number 1.712414 .4172931 2.21 0.027 1.062141 2.760804 riskfactors

-   Model 4: Observation subgroup, outcome=significant coronary disease,    primary predictor=sum (16:1, 18:1)

Logistic regression Number of obs = 62 LR chi2(2) = 19.31 Prob > chi2 =0.0001 Log likelihood = −17.737305 Pseudo R2 = 0.3525 AUC = 0.8885sig_cad Odds Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum 1.004249.0014328 2.97 0.003 1.001445 1.007061 (16:1 18:1) age 1.171344 .08276062.24 0.025 1.019866 1.34532

-   Model 5: Entire study cohort, outcome=significant coronary disease,    primary predictor=sum (16:1, 18:1, 18:3, 20:4, 20:5, 22:6)

Logistic regression Number of obs = 113 LR chi2(5) = 68.35 Prob > chi2 =0.0000 Log likelihood = −43.151639 Pseudo R2 = 0.4420 AUC = 0.9056sig_cad Odds Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum(significant CE) 1.002624 .0007152 3.67 0.000 1.001223 1.004027 age1.154735 .0419362 3.96 0.000 1.075399 1.239924 white 4.364957 3.2678441.97 0.049 1.006293 18.93371 male 4.051486 2.613006 2.17 0.030 1.14454214.34158 framingham 1.69183 .4264504 2.09 0.037 1.032283 2.772774

-   Model 6: Observation subgroup, outcome=significant coronary disease,    primary predictor=sum (16:1, 18:1, 18:3, 20:4, 20:5, 22:6)

Logistic regression Number of obs = 62 LR chi2(2) = 19.58 Prob > chi2 =0.0001 Log likelihood = −17.602642 Pseudo R2 = 0.3574 AUC = 0.8827sig_cad Odds Ratio Std. Err. z P > |z| [95% Conf. Interval] Sum 1.002625.0009186 2.86 0.004 1.000826 1.004427 (signifi- cant CE) age 1.178033.0876034 2.20 0.028 1.01826 1.362876Formula for model 1In(Probability any stenosis/1−probability anystenosis)=−17.2+0.692(number of risk factors)+0.0065(concentration CE16:1+concentration 18:1)+0.216(age)+1.12(gender*)* gender=1 if male, 2 if femaleFormula for model 3In(Probability significant stenosis/1−probability significantstenosis)=−11.97+0.538(number of risk factors)+0.0035(concentration CE16:1+concentration 18:1)+0.144(age)+0.657(gender*)* gender=1 if male, 2 if femaleIII. Discussion.

The results of this analysis suggest that plasma concentrations ofcholesteryl esters, the synthesis of which was predominately catalyzedvia ACAT2 are associated with coronary stenosis in patients with acutechest pain. The association was significant when examined in the entirestudy cohort consisting of patients with a wide spectrum of diseaseseverity, and remained significant when restricting the analysis to thesubgroup of emergency department patients with low-risk chest pain.

The background information suggesting that cholesteryl ester compositionmight matter in coronary artery atherosclerosis comes from numerousstudies done in animal models and in humans. Perhaps the firstobservations to directly establish a link came in 1997 when it was shownthat the cholesteryl ester secretion rate from livers in isolatedperfusion was predictive of the extent of coronary arteryatherosclerosis in the liver donor monkeys.(9) The acyl composition forcholesteryl esters made by the tissue esterifying enzyme ACAT waspredominantly oleate, and percentage of this CE was enhanced when thediet was enriched in oleate. The plasma enzyme LCAT contributes mostlycholesteryl linoleate. Subsequent studies in ARIC(21) and in Sweden(22)have confirmed that in humans, plasma cholesteryl oleate is positivelyassociated with carotid intimal medial thickness or mortality fromCardiovascular disease, respectively. Numerous studies in geneticallyengineered mice lacking the enzyme ACAT2 have also showed that markedreductions in cholesteryl oleate in plasma LDL occur together withreduced aortic atherosclerosis when ACAT2 is absent(23). Thus, aconvincing picture is available that a higher percentage of cholesteryloleate in plasma LDL cholesteryl esters is consistently associated withincreased atherosclerosis.

In order to be useful as a biomarker, CE measurements have to beobtainable. The measurements in this analysis were conducted in aresearch lab using a mass spectrometer. These instruments are relativelyexpensive but multiplexing an instrument use for more than one type ofanalyses would substantially reduce sample cost. Technical expertisewould require an individual that can dilute 5 to 10 μL of plasma orblood into a fixed volume of solvent. Mass spectrometer upkeep wouldrequire a service contract and an individual that could tune and checkthe instrument daily. Since most academic institutions have one or moremass spectrometer facilities, establishing this sort of facility wouldbe feasible. A typical cost for sample analysis in an academic massspectrometer facility is on the order of $30. If a modern electrospraymass spectrometer is installed the turn-around time would be minimal,0.5-1 h.

There exists a substantial need for more refined risk stratificationschemes to guide the evaluation of patients with acute chest pain.Existing clinical decision rules comprised of combinations oftraditional risk factors, biomarkers of necrosis and clinical variableshave proven insufficiently sensitive to exclude ACS.(13, 24, 25)Further, conventional risk factors for atherosclerosis have not provenuseful when evaluating patients with acute chest pain.(2, 6) Indistinction with prior efforts, we have chosen not to predict ACS, butrather to predict coronary stenosis. ACS consists of MI and unstableangina, both of which are most commonly the result of coronaryatherosclerotic disease. MI, by definition, is excluded with serialcardiac markers of necrosis. In contrast, unstable angina is defined bynon-elevated markers of necrosis and requires cardiac imaging for properdiagnosis. Reducing the need for cardiac imaging could improve health byreducing exposure to ionizing radiation using in the diagnostic testingprocess and could improve healthcare efficiency by reducing the numberof unnecessary imaging tests. Therefore, predicting a precursor forunstable angina, coronary atherosclerotic disease, could improve healthand increase healthcare efficiency. It should be noted that thisapproach could miss the relatively rare secondary causes of ACS if theyare not associated with myocardial necrosis, such as de-novo coronarythrombosis, coronary embolism, or coronary vasospasm.

The most clinically useful model in our analysis appears to be the sumof the plasma concentrations of CE 16:1 and CE 18:1, combined with age,gender, and the number of traditional coronary risk factors to predictthe presence of any coronary atherosclerosis in low risk patients. Ifvalidation of this model were successful, this measure could be used todetermine the need for coronary imaging or stress testing after theexclusion of MI. Our first examination in our low risk subgroup suggeststhat this approach could result in a meaningful reduction in imaging.Further prospective investigations should validate these findings,evaluate the potential clinical utility, refine appropriate cut-points,and examine model calibration.

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That which is claimed is:
 1. A method of measuring an amount of at leastone cholesteryl ester in a blood sample, comprising: combining saidblood sample with a salt and an alcohol to provide a dilute sample;introducing said dilute sample into a mass spectrometer; and thenmeasuring the amount of said at least one cholesteryl ester in saiddilute sample by mass spectrometry to thereby measure the amount of atleast one cholesteryl ester in said blood sample.
 2. The method of claim1, wherein: said alcohol is included in an amount sufficient tosolubilize said at least one cholesteryl ester in said sample; or saidsalt is included in an amount sufficient to form a metal adduct withsaid cholesteryl ester in said sample.
 3. The method of claim 1,wherein: said alcohol is selected from the group consisting of methanol,ethanol, and isopropanol; and/or said salt is selected from the groupconsisting of sodium formate, sodium acetate, ammonium formate, ammoniumacetate, lithium formate, lithium acetate, potassium formate, andpotassium acetate.
 4. The method of claim 1, wherein said blood sampleis selected from the group consisting of blood serum, blood plasma, orwhole blood.
 5. The method of claim 1, wherein said at least onecholesteryl ester (CE) is selected from the group consisting ofcholesteryl palmitate (CE 16:0), cholesteryl palmitoleate (CE 16:1),cholesteryl stearate (CE 18:0), cholesteryl oleate (CE 18:1),cholesteryl linoleate (CE 18:2), cholesteryl linolenate (CE 18:3),cholesteryl arachidonate (CE 20:4), cholesteryl eicosapentenoate (CE20:5), cholesteryl docosapentaenoate (CE 22:5), cholesteryldocosahexanoate (CE 22:6), and combinations thereof.
 6. The method ofclaim 1, further comprising adding an additional known cholesteryl esterin a known amount to said dilute sample as an internal standard.
 7. Amethod of measuring the amount of at least one cholesteryl ester in ablood sample, comprising: combining said blood sample with a salt and analcohol to provide a dilute sample; introducing said dilute sample intoa mass spectrometer; and then measuring the amount of said at least onecholesteryl ester in said dilute sample by mass spectrometry to therebymeasure the amount of at least one cholesteryl ester in said bloodsample wherein said alcohol is included in an amount sufficient tosolubilize said at least one cholesteryl ester in said sample; and saidsalt is included in an amount sufficient to form a metal adduct withsaid cholesteryl ester in said sample.
 8. The method of claim 7,wherein: said alcohol is selected from the group consisting of methanol,ethanol, and isopropanol; and/or said salt is selected from the groupconsisting of sodium formate, sodium acetate, ammonium formate, ammoniumacetate, lithium formate, lithium acetate, potassium formate, andpotassium acetate.
 9. The method of claim 7, wherein said blood sampleis selected from the group consisting of blood serum, blood plasma, andwhole blood.
 10. The method of claim 7, wherein said at least onecholesteryl ester (CE) is selected from the group consisting ofcholesteryl palmitate (CE 16:0), cholesteryl palmitoleate (CE 16:1),cholesteryl stearate (CE 18:0), cholesteryl oleate (CE 18:1),cholesteryl linoleate (CE 18:2), cholesteryl linolenate (CE 18:3),cholesteryl arachidonate (CE 20:4), cholesteryl eicosapentenoate (CE20:5), cholesteryl docosapentaenoate (CE 22:5), cholesteryldocosahexanoate (CE 22:6), and combinations thereof.
 11. The method ofclaim 7, wherein said blood sample is blood serum.
 12. The method ofclaim 7, wherein said blood sample is blood plasma.
 13. The method ofclaim 7, wherein said blood sample is whole blood.